CN111893119B

CN111893119B - Method for obtaining SCD1 gene editing goat embryo by using CRISPR/Cas9 system and microinjection

Info

Publication number: CN111893119B
Application number: CN202010814808.9A
Authority: CN
Inventors: 田慧彬; 牛慧敏; 罗军; 姚玮玮; 李聪; 耿亚楠; 雷安民; 史怀平
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2024-04-09
Anticipated expiration: 2040-08-13
Also published as: CN111893119A

Abstract

The invention discloses a method for obtaining SCD1 gene editing goat embryo by using CRISPR/Cas9 system and microinjection. In vitro transcription to obtain a CRISPR/Cas9 system, and microinjection of goat fertilized eggs is carried out by adopting the principle that two sgRNAs target the same target gene together; after the milk goats are subjected to synchronous estrus and superovulation treatment, embryo flushing is carried out 8-12 hours after the ewes are subjected to mating last time, namely about 46-50 hours after estrus begins, prokaryotic fertilized eggs are obtained, and microinjection is carried out in M199 culture solution containing 10% fetal bovine serum; the invention improves the development rate and the gene editing efficiency of the SCD1 gene editing goat embryo, and has good popularization and application prospect and economic value.

Description

Method for obtaining SCD1 gene editing goat embryo by using CRISPR/Cas9 system and microinjection

Technical Field

The invention belongs to the fields of genetic engineering and genetic modification, and relates to a goat embryo high-efficiency gene editing method established by utilizing a CRISPR/Cas9 system and a microinjection technology.

Background

The dairy goat industry is an important component of the modern dairy industry, and goat milk products are high in nutrition content, rich in short and medium chain fatty acids and various unsaturated fatty acids, and have unique nutritional values and health care functions (Clark and Garcia, 2017), so that optimization of the composition and content of goat milk fatty acids is necessary (Mahdi et al, 2018). The stearoyl-CoA desaturase (SCD 1) gene catalyzes the production of monounsaturated fatty acids, an important constituent of saturated fatty acids in sheep milk (Bernard et al 2005). The SCD1 gene knockout goat embryo can lay a foundation for living animal experiments, and provides a test animal material for researching the regulation and control effect of the SCD1 gene on the goat milk fatty acid, and has important significance.

CRISPR/Cas9 technology has become a main means for realizing transgenic animal production and molecular breeding, can efficiently and accurately edit specific loci of genome, such as deletion, insertion, repair and the like (Watakabe et al, 2018), and provides an effective way for directionally and accurately changing genetic materials and regulating animal genetic traits. Currently, CRISPR/Cas9 system mediated gene editing techniques have been widely used to construct animal models such as knockout mice and rats, and antitubercular transgenic cattle, anti-blue-ear pigs, lean pigs, and knockout down-converting goats, etc. have been successfully obtained using the gene editing techniques (Gao et al, 2017; wang et al, 2015; wu Tianwen, et al, 2017).

The microinjection technique is applied to CRISPR/Cas9 system mediated gene editing, which can significantly improve the gene editing efficiency (Qin et al, 2015), which can be as high as 100% in Drosophila embryos (Yu et al, 2013), but lower in mammalian embryos. In order to improve the editing efficiency, chinese patent CN106148416a cultures fertilized eggs of sgrnas and Cas9 mrnas for different subtypes of the Cyp gene in vitro by microinjection, and selects surviving fertilized eggs to transfer into recipient rats, and mating (selfing after crossing with wild type rats) is performed by using the produced rat offspring, so as to obtain homozygous rats with knockout target subtype, but for goats with long reproductive cycle and relatively insufficient fertility, it is difficult to obtain enough fertilized eggs with high homogeneity and good quality due to large individual difference. Also, in order to improve editing efficiency, chinese patent CN106957857a performs prokaryotic injection on two sgrnas and Cas9 mRNA designed for different or same exon regions of goat MSTN gene and FGF5 gene respectively to reduce miss rate, but the gene expression regulation and control pathway in the development period of mammal embryo is complex, and the increase of targeting site makes it more difficult to produce transgenic animals by using CRISPR/Cas9 system and microinjection, which mainly has the following problems: the embryo development rate of gene editing is low, and the birth rate of offspring is low.

Development of fertilized eggs in mammals (in the case of mice) can be defined as different Prokaryotic (PN) stages, namely PN1 to PN5. During PN1 and PN2, pre-replicated prokaryotes are smaller, localized to the embryo periphery after fertilization, and begin to migrate toward the embryo center during the G1 phase (10 h post fertilization); during PN3 and PN4, the prokaryotes undergo DNA replication, migrating further toward the center and toward each other during the S phase (10-16 h post fertilization); at PN5, the embryo is almost in G2 phase. The gene editing efficiency and the damage degree of fertilized eggs after injection are closely related to the Prokaryotic (PN) stage of microinjection selection, and researches report that when a mouse embryo is in the S stage of a cell cycle, a compound of Cas9 mRNA and sgRNA is introduced by utilizing the microinjection technology, and the gene editing is started and lasts for 12 to 24 hours after about 3 hours. However, in the case of goats, the limited number of fertilized eggs results in the impact of injected liquid on fertilized eggs during microinjection, and the difficulty in controlling the amount of injected liquid is high, which is more remarkable in the factors that restrict embryo survival and later development.

At present, no report exists on SCD1 gene editing of goat embryos by using CRISPR/Cas9 system and microinjection.

Disclosure of Invention

The invention aims to provide a method for obtaining an SCD1 gene editing goat embryo by using a CRISPR/Cas9 system and microinjection.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a method for obtaining goat gene editing embryos using CRISPR/Cas9 system and microinjection, comprising the steps of:

1) Obtaining a CRISPR/Cas9 system by in vitro transcription, the system comprising Cas9 mRNA and two sgrnas for co-targeting a goat SCD1 gene; the target sequences identified by the sgRNA are respectively positioned in the sequences of the goat SCD1 gene translation initiation part and the functional domain part;

2) And injecting the CRISPR/Cas9 system into a prokaryotic fertilized egg of a donor goat, transplanting the injected prokaryotic fertilized egg into a recipient goat body or placing the injected prokaryotic fertilized egg into a culture medium and continuing to develop to obtain the SCD1 gene editing goat embryo.

Preferably, the target sequence recognized by the sgRNA is located in the first exon and the third exon of the goat SCD1 gene.

Preferably, the target sequence recognized by the sgRNA (i.e., the coding sequence of the sgRNA, the sgRNA sequence) is:

5'-GGCCCACTTGCTGCAAGAGG-3' (6-25 bp of goat SCD1 Gene CDS)

5'-GGACCCCTGCTGTGATGCCC-3' (339-358 bp of goat SCD1 gene CDS).

Preferably, the step 1) further includes the steps of: the activity of sgrnas was detected using an in vitro cleavage assay.

Preferably, in the step 2), the ewes are subjected to synchronous estrus and superovulation treatment, then the estrus ewes are mated with the ram, and fertilized eggs are collected 8-12 hours after the last mating of the estrus ewes (i.e. about 46-50 hours after the start of estrus); in vitro injection was performed on the fertilized eggs using microinjection coupled to a microinjection apparatus.

Preferably, the injection parameters of the microinjection apparatus are set as follows: the injection pressure pi is 150-400hPa, the injection time t is 0.1-0.3s, and the pressure pc is maintained at 30-50hPa.

Preferably, the microinjection needle is drawn by a needle drawing instrument, and the drawing parameters are set as follows: heat=786-846, pull=90-110, variability=140-160, time=190-210.

Preferably, the concentration of Cas9 mRNA is 50-100 ng/. Mu.L and the concentration of sgRNA is 25-50 ng/. Mu.L.

A kit for obtaining goat gene editing embryos by using a CRISPR/Cas9 system and microinjection, which comprises an in vitro transcription template of the sgrnas (constructed in a corresponding cloning vector) and an in vitro transcription template of Cas9 mRNA.

The beneficial effects of the invention are as follows:

the invention utilizes the sgRNA of the goat SCD1 gene translation initiation part and the functional domain part obtained by in vitro transcription for microinjection, not only can provide more SCD1 gene editing embryos (blastula) to improve the birth rate of offspring, but also provides reliable technical basis for accelerating the breeding process of the milk goats with excellent milk production performance, and has good popularization and application prospects and economic values.

Furthermore, the method carries out injection on the obtained goat embryo in a certain prokaryotic stage according to the optimized microinjection parameters, and precisely controls (according to the injection parameters) the liquid injection quantity (1-2 pL) of the fertilized ovum by utilizing the microinjection needle (the caliber of which is not more than 1 mu m) manufactured by a needle pulling instrument, thereby ensuring the survival rate of the embryo after injection to the maximum extent.

Furthermore, the sgRNA aiming at the first exon and the third exon of the goat SCD1 gene and the in vitro transcription product of the Cas9 gene are injected together for microinjection, so that higher goat embryo SCD1 gene editing efficiency and higher development rate of a gene editing embryo (blastula) can be obtained.

Drawings

FIG. 1 is a schematic representation of the design of (Chr.26) SCD1 gene (SCD 1 locus) sgRNA located on chromosome 26 of goat.

FIG. 2 shows an in vitro transcription electrophoresis pattern of goat sgRNA; wherein: 9 represents the lane of sgRNA9, 16 represents the lane of sgRNA 16.

FIG. 3 is an in vitro transcriptional electrophoretogram of Cas9 mRNA.

FIG. 4 shows the results of in vitro cleavage activity assays; wherein: 9 represents the lane of sgRNA9, 16 represents the lane of sgRNA 16.

FIG. 5 is a schematic view of microinjection of fertilized goat eggs.

FIG. 6 shows the result of genomic sequence alignment of goat embryo SCD1 gene sgRNA before and after targeting; wherein: WT represents the result of partial sequencing of the SCD1 gene of the goat genome before targeting (which is strictly conserved as determined by alignment with the reference sequence), + represents the insertion, -represents the deletion, m represents the point mutation, and # represents the number of experimental fertilized eggs.

FIG. 7 is a flow chart of a method for establishing efficient editing of milk goat embryo genes based on microinjection in an embodiment of the invention.

Detailed Description

The invention will now be described in further detail with reference to the drawings and examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention.

Design of (one) sgRNA

Referring to FIG. 1, annealing primers (corresponding to sgRNA sequences sgRNA9, sgRNA16 as shown in Table 1) containing the sgRNA oligonucleotide sequences targeting the first exon E1 and the third exon E3 of the goat SCD1 gene and the reverse complement were synthesized according to the online site CHOPCHOP (http:// CHopchop. Cbu. Uib. No /), wherein the respective oligonucleotide sequence ends were added with BsaI cleavage site sequences. The synthesized single-stranded primers were subjected to DNA annealing and renaturation (see tables 2, 3) to form sgRNA double-stranded oligonucleotides with cohesive ends.

TABLE 1 goat SCD1 Gene sgRNA sequence and annealing primer

Note that: the locations of sgrnas in SCD1 gene CDS (GenBank: GU 947654.1) were: the sgRNA9 is between 6 and 25bp, and the sgRNA16 is between 339 and 358bp; only the target sequence is retained in the annealing primer, and PAM sequence at the target site is not included.

TABLE 2 sgRNA annealing System

TABLE 3 sgRNA annealing procedure

(two) in vitro transcription of sgRNA and Cas9 mRNA

The vector pUC57-sgRNA expression vector (Addgene 51132) for in vitro transcription of sgRNA was digested with BsaI overnight at 37 ℃ (Table 4), the linearized vector obtained after digestion was recovered, the double-stranded oligonucleotide of sgRNA was ligated with the linearized vector overnight at 16 ℃ (Table 5) using T4 DNA ligase, then Top10 E.coli competence (CB 104, beijing antenna) was transformed, single colony was cultured overnight in LB medium and plasmid was extracted through kanamycin resistance screening, and correctly constructed goat SCD1 gene sgRNA in vitro transcription template cloning vector pUC57-sgRNA (specifically pUC57-sgRNA9, pUC57-sgRNA16, according to the sequence of sgRNA) was obtained by sequencing.

TABLE 4 vector cleavage System

TABLE 5 connection System of sgRNA to linearization vector

The pUC57-sgRNA vector with correct sequence is used as a template, and the sgRNA in-vitro transcription template is obtained by PCR, and the primer sequences are shown in Table 6:

TABLE 6 PCR primers for amplifying in vitro transcription template T7-sgRNA

The PCR reaction system and the procedure are shown in Table 7:

TABLE 7 PCR reaction System and procedure for amplifying in vitro transcription template T7-sgRNA

The T7-sgRNA9 obtained by PCR comprises: t7 promoter, sgRNA9 sequence, and sgRNA scaffold;

the T7-sgRNA16 obtained by PCR comprises: a T7 promoter, an sgRNA16 sequence, and an sgRNA scaffold;

wherein the T7 promoter sequence is 5'-TAATACGACTCACTATAGG-3'; the sgRNA scaffold sequence is 5'-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3'.

And (3) cutting and recovering the PCR product after electrophoresis, recovering in-vitro transcription template DNA by using an Axygen gel recovery kit, adding RNAsecure Reagent into a recovered DNA system according to the ratio of 1:25, carrying out metal bath at 60 ℃ for 10min, recovering the PCR product again in an enzyme-free environment by using the Axygen PCR clean-up recovery kit, and removing RNase in the template.

The recovered DNA was purified as an in vitro transcription template for sgRNA, and the concentration of the recovered DNA was determined according to MEGAShortscript ^TM In vitro transcription is carried out according to Kit instruction, the dosage of template DNA is 800ng, the reaction is carried out for 4 hours at 37 ℃, DNase I is added according to the instruction after transcription, the template DNA is removed, and then the template DNA is removed according to MEGAclear ^TM Kit instructions purified sgrnas. The quality of the purified sgRNA is detected by 180V and 2% agarose gel electrophoresis (see figure 2, the sgRNA obtained by in vitro transcription has clear bands and no tailing, which shows higher purity, and can be used for fertilized egg microinjection), and the quality of the RNA is measured, split-packed and stored at-80 ℃.

Cas9 expression plasmid pST1374-NLS-flag-linker-Cas9 (Addgene 44758) was digested with restriction enzyme AgeI overnight at 37 ℃.

TABLE 8-1 vector cleavage System

RNAsecure Reagent is added into the reaction liquid after enzyme digestion according to the proportion of 1:25, metal bath is carried out for 10min at 60 ℃, a PCR Purification Kit recovery kit is used for recovering linearization vector and removing RNase in an enzyme-free environment, and the linearization vector is used as an in vitro transcription template of Cas9 mRNA, and the template DNA dosage is 800ng. MmessageMmachine using Cas9 in vitro transcription kit ^TM T7 Ultra Kit for in vitro transcription and tailing according to the Kit instructions(see table 8-2) and purified and recovered using the RNeasy Mini Kit to obtain the tailed Cas9 mRNA.

TABLE 8-2 Cas9 mRNA tailing System

Note that: the components of the system are gently mixed, placed at 37 ℃ and incubated for 30-45min, and placed in ice after the completion of the incubation.

Referring to fig. 3, cas9 mRNA obtained by in vitro transcription has a greater post-tailed band than a pre-tailed band, indicating that mRNA was successfully tailed with a, and is available for translation to form Cas9 protein.

(III) in vitro cleavage Activity experiment

To verify that in vitro transcribed sgrnas can bind correctly to Cas9 protein and cleave the target sequence, the transcribed sgrnas are used to incubate with DNA fragments comprising the target site of the sgrnas and Cas9 protein, as described in detail below.

First, DNA containing the sgRNA target site was obtained by PCR using wild-type genomic DNA as a template and using amplification primers for different exons of the SCD1 gene (Table 9). The reaction system and procedure of PCR can be referred to in Table 7.

TABLE 9 goat SCD1 Gene exon amplification primers

Next, after the PCR product was added to RNA secure Reagent, the RNase was removed by a metal bath at 60℃for 10 minutes, and recovered in an enzyme-free environment using PCR Purification Kit recovery kit. Using the in vitro cleavage assay system (Table 10), the activity of transcribed sgRNA was examined to determine the sgRNA for microinjection.

TABLE 10 in vitro cleavage test System

In the in vitro cleavage test, the mixed sample prepared according to the above test system was incubated at 37℃for 30min, and DNA was recovered using a DNA recovery kit to remove other impurities such as proteins and buffers. In vitro cleavage of the target DNA fragment (i.e., DNA comprising the sgRNA target site) by Cas9 protein (i.e., cas9 nuclease) under the guide of the experimental sgrnas was detected by 1% agarose gel electrophoresis.

Referring to FIG. 4, the corresponding sgRNAs obtained after in vitro transcription of T7-sgRNA9 and T7-sgRNA16 can form a complex with Cas9 protein in vitro and combine with the complex to cut on a target DNA fragment, and the cut DNA is broken into small fragments, so that the sgRNAs 9 and 16 are proved to have activity and can be used for microinjection.

(IV) microinjection of fertilized eggs

Referring to fig. 7, the invention establishes a flow of a method for efficiently editing milk goat embryo genes based on microinjection, which is specifically as follows.

1. Gene editing injection

Two sgrnas (specifically, T7-sgRNA9, T7-sgRNA16 in vitro transcripts) were mixed together with the tailed Cas9 mRNA in nuclease-free water for injection. The resulting injections were mixed to have a concentration of Cas9 mRNA of 50 ng/. Mu.l and a concentration of 25 ng/. Mu.l per sgRNA.

2. Acquisition of milk goats under test

The milk goats used in the test are the West NongSacan milk goats of the stock farm of Sacan milk goats of the university of North agriculture and forestry science and technology in the demonstration area of Shaanxi Yang Ling, and the test is performed for 2019 and 9 months.

3. Synchronous estrus and superovulation

The donor ewe and recipient ewe placed vaginal CIDR plugs on the same day.

And (3) carrying out synchronous estrus and superovulation treatment on the donor ewes: donor ewes were injected with 4mL of FSH 19:00pm on day 12 where CIDR plugs were placed, followed by three days of FSH injections of 7:00am and 19:00pm daily at doses of 3mL, 2mL, 1.5mL, 1mL, respectively, with 0.1mg PG being injected the last time the FSH injection, followed by thrombolysis. Mating the oestrus ewes.

And (3) carrying out synchronous estrus treatment on the recipient ewes: recipient ewes were injected with 330 units of PMSG at 19:00PM on day 12 with CIDR bolts and with 7:00AM thrombolytic on day 15, with 0.1mg PG.

4. Mating and collecting fertilized eggs

And determining embryo flushing time according to the starting time and the ending time of estrus after the estrus ewes are in the stud with the ram. After the last mating of the ewes (mating, indicating that the ewes are in oestrus; mating is not accepted, indicating that oestrus is over) 8-12 hours (namely about 46-50 hours after oestrus begins), oviduct embryo punching is carried out to obtain fertilized eggs of the dairy goats. Fertilized eggs are placed in M199 culture solution containing 5% fetal calf serum, and embryo detection results show that the obtained fertilized eggs are in a prokaryotic stage.

5. Microinjection of fertilized eggs from milk goats

Referring to fig. 5, microinjection was performed in M199 medium containing 10% fetal bovine serum using a FemtoJet microinjection apparatus.

Before injection, a microinjection needle suitable for goat fertilized eggs is drawn by using a Micropipette Puller P-97 type needle drawing instrument, and parameters of the needle drawing instrument are set as follows: heat=816, pull=100, variability=150, time=200. The optimal injection parameters were determined as follows: injection pressure pi=400 hPa, injection time ti=0.3 s, maintenance pressure pc=30 hPa.

After microinjection is finished, fertilized eggs are observed under a microscope, the number of embryo deaths caused by microinjection is counted, and the result shows that the embryo survival rate can reach 82.7+/-2.9% after the comparison with the total number of fertilized eggs before injection.

6. Milk goat embryo SCD1 gene editing efficiency detection

And (3) injecting sgRNA aiming at two exons of the SCD1 gene of the genome of the dairy goat and the Cas9 mRNA after tailing into fertilized eggs of the goat together under the optimal injection parameters, placing the fertilized eggs after injection into a 37 ℃ incubator for half an hour, quickly transporting the surviving fertilized eggs to an embryo transplantation operating room, and transplanting the fertilized eggs to oviducts of a female goat of a recipient. And (5) normal feeding management of the transplanted ewes and monitoring of health conditions. Meanwhile, in order to facilitate observation of embryo development, the in vitro culture of part of surviving fertilized eggs is reserved, single embryos are collected when the embryos develop to the blastula stage, genome extraction is carried out by using a single Cell genome amplification Kit REPLI-gSingle Cell Kit (Qiagen, 150343), then PCR amplification and sequencing are carried out on gene editing sites, and the gene editing efficiency of corresponding sgRNA at the embryo level of the milk goat is analyzed and calculated according to the result.

In vitro culture: some of the surviving fertilized eggs were transferred into the G1 embryo culture medium, washed three times, and placed in 25. Mu.L droplets for culturing (surface-coated with paraffin oil, 10-15 embryos in each droplet). After in vitro culture for 24 hours, the embryo develops to a two-cell stage, the embryo is transferred into a G2 embryo culture solution rich in glucose for continuous culture until the 7 th day, and the embryo developed to a blastula stage is counted. The results showed that the average number of milk goat embryos developed to blastula in each droplet was 7.

Referring to FIG. 6, the sgRNA/Cas9 mRNA injected into fertilized eggs of the present invention plays a role in efficiently editing a target gene (SCD 1 gene) in milk goat embryos, and meanwhile, the embryo (blastula) obtained after microinjection of two sgRNAs has a gene editing efficiency of 35% (7/20), wherein the sgRNA9 gene editing efficiency is 20% (4/20) and the sgRNA16 gene editing efficiency is 30% (6/20). Thus, co-injection of two sgrnas can increase the gene editing efficiency of fertilized eggs.

(fifth) Experimental controls and supplementary notes

1. Design description of sgrnas

The invention screens the sgRNA (sgRNA 9, sgRNA 16) with highest efficiency according to the oocyte level gene editing result, and then is used for goat fertilized eggs.

2. For determining the collection time of fertilized eggs in the procaryotic stage

After the estrus of the ewe is observed, the estrus of the ewe is tested every 8 hours, the estrus of the ewe is monitored for 3 times within 24 hours, and the starting time and the ending time of the estrus of the ewe are accurately mastered. Considering both aspects, i.e. monitoring the start and end of oestrus simultaneously, and determining that collection of fertilized eggs in the pro-nuclear period is performed 8-12 hours after the last mating of the ewe (i.e. about 46-50 hours after the start of oestrus) is more accurate than the prior art, which generally determines the collection time of fertilized eggs in the pro-nuclear period based on the start time of oestrus (particularly for determining injection time).

3. Effects of injection on embryo development

In the prior art, the development rate of blastula after microinjection of fertilized eggs of mice can reach 24.78% (28/113; xu Wenhao, 2018). The development rate of the blastocyst of the milk goat embryo after microinjection can reach 70% (7/10).

In a word, in the invention, in the process of carrying out goat embryo gene editing by utilizing the CRISPR/Cas9 system and microinjection, the survival rate of fertilized eggs after injection is improved and a higher embryo development rate is obtained by controlling and optimizing SCD1 gene target sites, microinjection time and parameters.

<110> university of agriculture and forestry science and technology in northwest

<120> method for obtaining SCD1 Gene-edited goat embryo Using CRISPR/Cas9 System and microinjection

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Claims

1. A method for obtaining goat gene editing embryos by using a CRISPR/Cas9 system and microinjection, which is characterized in that: the method comprises the following steps:

1) CRISPR/Cas9 system obtained by in vitro transcription, comprising Cas9 mRNA and use for targeting goatsSCD1Two sgrnas of a gene;

the target sequence recognized by the sgRNA is as follows:

5’-GGCCCACTTGCTGCAAGAGG-3’

and

5’-GGACCCCTGCTGTGATGCCC-3’；

2) Injecting the CRISPR/Cas9 system into a prokaryotic fertilized egg of a donor goat, placing the injected prokaryotic fertilized egg into a culture medium and continuing to develop to obtainSCD1Gene editing goat embryo;

in the step 2), fertilized eggs collected 8-12 hours after the last mating or 46-50 hours after the beginning of the estrus are subjected to in-vitro microinjection, the caliber of a microinjection needle is not more than 1 mu m, the liquid injection amount of the fertilized eggs is 1-2pL, the concentration of Cas9 mRNA is 50-100 ng/mu L, and the concentration of each sgRNA is 25-50 ng/mu L;

the microinjection needle used for microinjection is formed by drawing by a needle drawing instrument, and drawing parameters are set as follows: heat=786-846, pull=90-110, variability=140-160, time=190-210;

the injection parameters of the microinjection are set as follows: the injection pressure pi is 150-400hPa, the injection time t is 0.1-0.3s, and the pressure pc is maintained at 30-50hPa.